Systemic or localized forms of bone loss are caused by a variety of diseases or conditions, including aging, and the resulting osteopenia leads to numerous clinical manifestations including increased incidence of fracture. The identification of novel anabolic agents in bone, and perhaps even more importantly, gaining insights into their mechanisms of action, are subjects of intense clinical interest. CTGF has recently emerged as an important factor in osteogenesis and we recently demonstrated that the bone growth factor, TGF-21, is a potent inducer of CTGF expression and that CTGF is a downstream mediator of TGF-21-induced ECM production in osteoblasts. The mechanisms that govern CTGF induction by TGF-21 are cell type specific and little is known about this mechanism in osteoblasts. We present preliminary data that demonstrates for the first time the unique requirement of two promoter motifs the Transforming growth factor-beta response element (TRE) and the Smad binding element (SBE) for CTGF promoter trans-activation. These results led to our hypothesis that in osteoblasts the TRE and SBE function as cognate motifs and that both elements cooperate to achieve induction of CTGF expression by TGF-21. The identities of nuclear proteins that bind to the SBE, the TRE, or both have not been investigated in osteoblasts, and we propose in Aim 1 to investigate these proteins with a focus on proteins that require both motifs, since they may be unique to osteoblasts. In our preliminary data, we demonstrated the requirement of Smad and Erk signaling for TGF-21 up-regulation of CTGF in osteoblasts and we also demonstrate that expression of the transcription factor Ets-1 synergizes with TGF-21 to induce CTGF promoter activation in osteoblasts. MAPKs such as Erk can modulate the Smad pathway through directly mediating the phosphorylation (activation) of Smads or indirectly through activation/ inactivation of required nuclear transcription factors that mediate Smad DNA binding. Our preliminary data demonstrates that Erk signaling is not required directly for Smad activation but is required for transcriptional complex formation on the SBE and TRE on the CTGF promoter, demonstrating that Erk signaling is important for SBE and TRE trans-activation. Ets-1 is known to function as a downstream mediator of Erk signaling in some cell types and recently Ets-1 has been shown to function in a combinatorial manner with Smad proteins. In Aim 2, we propose to investigate the role of Erk and Ets-1 signaling for CTGF induction by TGF-21 in osteoblasts, specifically if Erk translocates to the nucleus and is required for Smad trans-activation of the SBE, the role of Ets-1 in TGF-21 induced CTGF, and if Ets-1 is a downstream effector for Erk mediated CTGF induction by TGF-21. Identification and characterization of molecular mechanisms that work to promote osteoblast differentiation and function may be helpful in developing new therapeutic strategies to selectively enhance bone formation in patients with significant bone loss. PUBLIC HEALTH RELEVANCE: Osteoporosis is a major health care problem since approximately 10 million people over the age of 50 have been diagnosed with the disease and 33.6 million more are estimated to have low bone mass (osteopenia). Low bone mass is accompanied by an increased incidence of fracture, and it is estimated that the direct health care costs from fractures related to osteopenia (hospitalizations, ER visits, physician visits, etc.) ranges from $12-$18 billion annually. CTGF is a novel growth factor in bone and the proposed studies will generate new information regarding its regulation by TGF-21 in osteoblasts. Developing an understanding of the growth control mechanisms in osteoblasts, and particularly the identification and characterization of the mechanisms of TGF-21 and CTGF in regulating osteoblast differentiation and function, will help identify new therapeutic targets to enhance bone formation in patients with clinically significant bone loss.